Effects of Drugs Formerly Proposed for COVID-19 Treatment on Connexin43 Hemichannels

Abstract

Connexin43 (Cx43) hemichannels form a pathway for cellular communication between the cell and its extracellular environment. Under pathological conditions, Cx43 hemichannels release adenosine triphosphate (ATP), which triggers inflammation. Over the past two years, azithromycin, chloroquine, dexamethasone, favipiravir, hydroxychloroquine, lopinavir, remdesivir, ribavirin, and ritonavir have been proposed as drugs for the treatment of the coronavirus disease 2019 (COVID-19), which is associated with prominent systemic inflammation. The current study aimed to investigate if Cx43 hemichannels, being key players in inflammation, could be affected by these drugs which were formerly designated as COVID-19 drugs. For this purpose, Cx43-transduced cells were exposed to these drugs. The effects on Cx43 hemichannel activity were assessed by measuring extracellular ATP release, while the effects at the transcriptional and translational levels were monitored by means of real-time quantitative reverse transcriptase polymerase chain reaction analysis and immunoblot analysis, respectively. Exposure to lopinavir and ritonavir combined (4:1 ratio), as well as to remdesivir, reduced Cx43 mRNA levels. None of the tested drugs affected Cx43 protein expression.

Keywords: COVID-19; cellular communication; connexin43; drug; hemichannel.

Figure 1

Concentration–response curves for the determination of the CC₁₀ after 24 h of drug exposure of transduced Dubai camel cells overexpressing human Cx43 (DUBCA-hCx43). A sigmoidal curve was fitted by means of non-linear regression using GraphPad^® Prism to determine the CC₁₀ value. Data are expressed as mean ± standard deviation (N = 4, n = 3).

Figure 2

Effects of the drug panel on Cx43 hemichannel activity of transduced human embryonic kidney cells overexpressing human Cx43 (HEK-hCx43). The released amount of ATP of HEK-hCx43 cells exposed for 30 min to azithromycin, chloroquine, dexamethasone, favipiravir, hydroxychloroquine, lopinavir, remdesivir, ribavirin, ritonavir, and the combination of lopinavir and ritonavir, was measured. A buffer with normal calcium levels (NC) was used to mimic physiological conditions in which the hemichannels are typically closed. A buffer without divalent ions (DF) was used to open Cx43 hemichannels, while carbenoxolone (Cbx) dissolved in DF buffer was used as a Cx43 hemichannel inhibitor. Significant differences between the test conditions and the DF buffer were calculated with a parametric one-way analysis of variance or a non-parametric Kruskal–Wallis test followed by a Dunnett’s or Dunn’s multiple comparison test, respectively, depending on the distribution (i.e., Shapiro-Wilk normality test). Data are expressed as mean ± standard deviation with * p ≤ 0.05 ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001. (N = 4, n = 3) for azithromycin, chloroquine, dexamethasone, favipiravir, hydroxychloroquine, lopinavir, ribavirin and ritonavir. (N = 4; n = 4) for remdesivir. (N = 4; n = 5) for the combination of lopinavir and ritonavir.

Figure 3

Cx43 protein expression after 24 h of drug exposure of transduced Dubai camel cells overexpressing human Cx43. Both the phosphorylated (P1 and P2) and non-phosphorylated (NP) variants could be detected. Signals of Cx43 were normalized against total protein loading and expressed as relative alterations compared to untreated controls (dashed line), using Image Lab software. Statistical analysis was performed using a parametric one-way analysis of variance or non-parametric Kruskal–Wallis test in combination with a Dunnett’s or Dunn’s test, depending on the normality of the data. Data are expressed as mean ± standard deviation (N = 1, n = 3).

Figure 4

Cx43 mRNA expression after 24 h of exposure of transduced Dubai camel cells overexpressing human Cx43 to the drugs. mRNA expression levels were measured using RT-qPCR. Data were analyzed with the Pfaffl method and normalized against untreated controls (dashed line) [50]. Statistical analysis was performed using a parametric one-way analysis of variance or non-parametric Kruskal-Wallis test in combination with a Dunnett’s or Dunn’s test, respectively, depending on the normality of the data (i.e., Shapiro–Wilk normality test). Data are expressed as mean ± standard deviation with * p ≤ 0.05 and **** p ≤ 0.0001 (N = 3, n = 3).